Compositions and Methods for Wound Treatment

ABSTRACT

A composition for treating a wound includes graphene oxide (GO) and hyaluronic acid (HA) that are covalently linked, XAV 939 , and water. The composition can also include a surfactant, such as PEG. The composition can be topically administered to a subject to treat a wound of the subject. Methods of treating a wound using the composition are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/569,025, filed Oct. 6, 2017, and to U.S. Provisional Application No.62/684,439, filed Jun. 13, 2018, the disclosure of each of which isincorporated by reference herein in its entirety.

BACKGROUND

The invention described herein relates to compositions and methods forwound treatment, and in particular, the prevention and/or reduction ofscar formation during wound healing.

Wound healing is a complex process, involving an inflammation phase, agranulation tissue formation phase, and a tissue remodeling phase. Theseevents are triggered by cytokines and growth factors that are releasedat the site of injury. Many factors can complicate or interfere withnormal adequate wound healing. Chronic wounds, such as diabetic footulcers, venous leg ulcers, and pressure ulcers are particularlytroublesome and challenging to treat.

A scar is the mark left in the skin by new connective tissue thatreplaces tissue which has been injured. Scarring in the skin aftertrauma, surgery, burn or sports injury can be a medical problem,resulting in loss of function, restriction of tissue movement andadverse psychological effects. Skin fibrosis, an irreversiblepathological process that causes a loss of normal tissue structure andorgan function, is associated with scarring. While the pathways andprocesses underlying scar formation have been better understood in therecent years, no effective therapeutic approaches for scar managementare available, and there are no prescription drugs for the prevention ortreatment of dermal scarring.

Currently available techniques for treating scarring (e.g., siliconesheeting (pressure therapy), topical ointments, resurfacing, peel,dermabrasion, lasers, cryosurgery, bleomycin and 5-fluorouracilinjection, excision (revision surgery, radiotherapy), reconstructionpossibly with skin grafts, flaps, etc.) focus on improving theaesthetics of existing scars, and have issues related to scarringrecurrence and side-effects, such as dermal atrophy andhypopigmentation.

The Wnt pathway has been recently shown to play a key role in dermalfibrosis and scarring. The Wnt pathway is an evolutionary conservedpathway that regulates crucial aspects of cell fate determination, cellpolarity, cell migration, neural patterning, and organogenesis duringembryonic development. This pathway is instrumental in ensuring propertissue development in embryos and tissue maintenance in adults. Wntsignaling is involved at the beginning stages of skin development.Following gastrulation, embryonic cells of the ectoderm and the mesodermdifferentiate to form the epidermis and dermis, respectively.

Although there are at least three distinct Wnt signaling pathwaysinvolved in the signal transduction process, the canonical (or β-catenindependent) Wnt pathway is the most understood. β-Catenin is the keyeffector molecule resulting from the signaling of the canonical Wntpathway, and its protein levels are regulated through a “destructioncomplex”. In the absence of a Wnt signal, the transcriptional activatorβ-catenin is actively degraded in the cell by the actions of a proteincomplex, designated the “destruction complex”. Within this complex,Axin-1 and -2 with adenomatous polypsis coli form a scaffold thatfacilitates β-catenin phosphorylation by casein-kinase 19a and glycogensynthase kinase 3β. Phosphorylated β-catenin is recognized andubiquitinylated, resulting in its proteosomal degradation. Tankryase Iand II (TNK1 and 2) are poly(ADP-ribose) polymerases (PARPs) thatfunction to parsylate and destabilize Axin-1 and -2 proteins, thusdestabilizing the β-catenin destruction complex. Once the destructioncomplex is destabilized, this allows β-catenin to be dephosphorylated,and subsequently stabilized and allowed to accumulate in the cytoplasmand enter the cell nucleus, where it interacts with members of theTcf/Lef family. β-catenin converts the Tcf proteins into potenttranscriptional activators by recruiting co-activator proteins, thusensuring efficient activation of Wnt target genes. The Wnt pathway, onceactivated by the Wnt family of natural ligands, upregulates TNK1 and 2to help destabilize the destruction complex. Studies have shown thatTNK1 and 2 are critical regulators of canonical Wnt signaling.

Canonical Wnt signaling is over-activated in a variety of tumors whereit plays a central role in cell growth and metastasis. In addition, theWnt pathway has been shown to regulate cell proliferation in the adultepidermis, indirectly impacting the rate and extent of skin woundhealing and fibrosis or scarring. Further, the Wnt/β-catenin pathway hasbeen shown to cause overstimulation of dermal fibroblasts, which cangive rise to myofibroblasts. Myofibroblasts are endowed with contractilefunction, which allows them to play a role in extracellular matrix (ECM)fibers to close open wounds. Overexpression of myofibroblasts causesexcess collagen and ECM protein secretion, which in turn causes fibrosisand scarring. Sustained β-catenin activity in dermal fibroblastspromotes fibrosis by upregulating expression of ECM protein-codinggenes. β-Catenin levels have been shown to regulate wound size andmediate the effect of TGF-β in cutaneous healing. The Wnt/β-cateninpathway has been shown to be upregulated in hypertrophic scars andkeloid fibroblasts. Thus, the Wnt/β-catenin pathway induces β-cateninsignaling in cutaneous mesenchymal cells, leading to their activationand induction of a sustained fibrotic response.

TNK1 and 2 have been shown to be druggable targets for modulation of theWnt/β-catenin pathway. However, recently it has been demonstrated thatsystemic inhibition of TNK1 and 2 can cause intestinal toxicity due toinhibition of intestinal crypt cell renewal, a process primarily drivenby the Wnt/β-catenin pathway.

XAV939 is a small molecule that selectively inhibitsWnt/β-catenin-mediated transcription through TNK 1 and 2 inhibition withan IC50 of 11 nM/4 nM in cell-free assays, regulates axin levels, anddoes not affect CRE, NF-κB, or TGF-β. Recently, topical application ofXAV939 in a mouse ear punch assay demonstrated that XAV939 significantlyincreased rate of wound closure with reduced fibrosis (scarring).However, XAV939 was dissolved in DMSO and used only as a “research tool”compound due to its very low aqueous solubility (<1 mg/mL). The problemwith this approach is that humans cannot tolerate the use of DMSO. Asoluble form of XAV939 suitable for humans is required for practical andmedical use.

SUMMARY OF THE INVENTION

In one aspect of the present invention, a composition for treating awound is provided. The composition comprises: a matrix componentcomprising graphene oxide (GO) and hyaluronic acid (HA), wherein the GOand HA are covalently linked via a linker; XAV939; and water. Thecovalently linked GO and HA is also referred to herein as GO-HA or GO-HAconjugate. The composition can be in the form of a suspension, where theGO-HA can be present in the forms of dispersed microparticles suspendedin the water.

In some embodiments, the composition further comprises a surfactant,which can be a polyethylene glycol (PEG). The PEG can have a molecularweight of about 200 to about 400 Daltons. The PEG can be in an amount offrom about 0.1 wt % to about 20 wt % of the total composition.

In some embodiments, the composition further comprises a thickener,which for example, can be hydroxypropyl cellulose (HPC).

In some embodiments, the linker linking the GO and HA includes 2-25carbons. In some embodiments, the linker can be straight-chained (orlinear). In other embodiments, the linker can be branched. In someembodiments, the linker comprises a linear alkylene —C_(m)H_(2m)— unitwhere m can be from 1 to 20. In some other embodiments, the linker cancomprise one or more heteroatoms. For example, the linker can includeone or more —CH₂CH₂O— units. In certain embodiments, the linkercomprises —R^(x)—R^(S)—R^(y)—, wherein R^(x) and R^(y) are eachindependently selected from the group consisting of —CO—, —COO—, —NH—,—NH—NH—, —NH—NH—CO—, —CS—, —S—, —O—, and wherein R^(S) is anunsubstituted or substituted linear alkylene group having 1-40, or 2-20backbone carbons. In specific embodiments, R^(x) and R^(y) are each—NH—NH—CO—.

In some embodiments, the weight ratio of XAV939 to GO-HA can be fromabout 1:100 to about 100:1, for example, from about 1:2 to about 2:1. Insome embodiments, XAV939 constitutes from about 0.001 wt % to about 5 wt% of the total composition. In certain embodiments, the GO-HAconstitutes from 0.001 wt % to about 5 wt % of the total composition.

In another aspect, a medical device is provided. The medical deviceincludes a substrate; and the composition(s) described herein which isapplied on the substrate. The substrate can be a patch, a pad, a suture,a gauze, a tape, or a bandage.

In a further aspect, a method of treating a cutaneous wound in a subject(a human or non-human animal, such as a mammal) by contacting the woundwith an effective amount of the composition(s) as described herein, isprovided. The wound can be a surgical wound or a burn. In someembodiments, the wound can be a chronic wound such as an ulcer. In someembodiments, the method further comprises delivering a second woundmedication to the subject, the second wound medication comprising one ormore of: corticosteroid, a cytotoxic drug, an antibiotic, an antiseptic,nicotine, an anti-platelet drug, an NSAID, colchicine, ananti-coagulant, a vasoconstricting drug or an immunosuppressive, agrowth factor, an antibody, a protease, a protease inhibitor, anantibacterial peptide, an adhesive peptide, a hemostatic agent, livingcells, honey, or nitric oxide.

In a further aspect, a method of preparing a GO-HA conjugate isprovided. The method includes: modifying GO by converting at least someof the benzoxylic acid groups of the GO to terminal aliphatic carboxylicacid groups; derivatizing HA by reacting HA with a reagent having dualfunctional groups reactive to the terminal aliphatic carboxylic acidgroups, the dual functional groups intervened by a spacer group; andreacting the modified GO and the derivatized HA to form the GO-HAconjugate. In some embodiments, the spacer group can comprise a linearalkylene having 2-20 backbone carbons. In some embodiments, the reagentfor derivatizing HA is a dihydrazide. In some embodiments, in the GO-HAconjugate, the weight ratio of GO:HA is from about 1:1 to about 1:20, orfrom about 1:6 to about 1:10.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot showing the effect of a composition of the presentinvention on scar reduction related to wound healing of an animal modelas compared to the effect of a vehicle composition.

FIG. 2 is a plot showing the effect of a composition of the presentinvention on the wound healing of an animal model as compared to theeffect of a vehicle composition.

DETAILED DESCRIPTION

In one aspect of the invention, a composition for treating a wound isprovided, which includes: a matrix component comprising a conjugate ofgraphene oxide (GO) and hyaluronic acid (HA) where GO and HA arecovalently linked via a linker; XAV939; and water. The covalently-linkedGO and HA is also referred to herein as GO-HA conjugate or simply GO-HA.

XAV939 is a potent tankyrase inhibitor, with a chemical name3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one.The structure of XAV939 is shown below:

Graphene oxide (GO) as used herein refers to an oxidized form ofgraphene, which is a single layer form of graphite. GO can be obtainedby treating graphite with strong oxidizers. GO contains carbon, oxygen,and hydrogen in various amounts, depending on how it is made. It canhave several hundreds of nanometers, up to several micrometers, itsplanar direction, and about 0.7-1.2 nm in thickness. GO can includevarious oxygen containing moieties, such as oxygen epoxide groups,carboxylic acid (—COOH), phenol, etc., when prepared using sulphuricacid (e.g. Hummers method). An example GO structure is shown below.

Hyaluronic acid (HA) is an anionic, highly hydrophilic, non-sulfatedglycosaminoglycan, occurring naturally throughout the human body. It canbe several thousands of carbohydrate units long, and can bind to watergiving it a gel of stiff viscous quality. An example structure of HA isprovided below:

In the composition of present invention, the GO and HA are covalentlylinked to form a matrix component (or a carrier), which can serve tosolubilize XAV939 as well as providing other simultaneous benefits towound healing. The covalent linking can be accomplished by using alinker (or linker moiety). In some embodiments, the linker can include2-25 carbons. In some embodiments, the linker is linear. In otherembodiments, the linker is branched. The linker can be saturated orunsaturated.

In some embodiments, the linker can comprise a C₂-C₂₅ alkylene group,where the carbons and hydrogens in the alkylene group can be substitutedby oxygen or other atoms or groups such as hydroxy, carboxy, amino,alkyl, alkoxy, alkenyl, alkynyl, nitro, etc. In some embodiments, thelinker can comprise one or more —CH₂CH₂O— units.

In some embodiments, the linker comprises —R^(x)—R^(S)—R^(y)—, whereinR^(x) and R^(y) are each independently selected from the groupconsisting of —CO—, —COO—, —NH—, —NH—NH—, —NH—NH—CO—, —CS—, —S—, —O—,and wherein R^(S) (which is also referred to as the spacer group in thisapplication) can be an unsubstituted or substituted, saturated orunsaturated linear alkylene group having 2-20 backbone carbons. Inparticular embodiments, both R^(x) and R^(y) are *—NH—NH—CO— (* denotingthe ends of the linker distal to R^(S)).

In some embodiments of the composition, the weight ratio of XAV939 toGO-HA can be from about 1:100 to 100:1, e.g., from about 1:2 to about2:1. In some embodiments, in the GO-HA conjugate, the weight ratio ofGO:HA can be from about 1:1 to about 1:20, or from about 1:6 to about1:10.

In general, the composition overall can appear as a slightly dark orblack viscous liquid. XAV939 is evenly dispersed in the viscoussuspension, which is stable at room temperature for months. In someembodiments, the composition further comprises a surfactant thatenhances mixability or solubility of hydrophobic substances in water. Insome examples, the surfactant can be a non-ionic hydrophilic materialsuch as polyethylene glycol (PEG). The PEG can have a number-averagedmolecular weight of from about 100 to about 10,000 Daltons, or about 200to about 4000 Daltons, e.g., from about 200 to about 1000, from about200 to about 800, from about 200 to about 500, from about 200 to about400, from about 300 to about 400, from about 350 to about 450, about200, about 250, about 300, about 350, about 400, about 450, about 500,about 550, about 600, about 650, about 700, about 750, about 800, about850, about 900, about 950, about 1000 Daltons, etc. In some embodiments,the PEG can be present in the composition in an amount of from about 0.1to about 20 wt % of that of the total composition. For example, the PEGcan be from about 0.2 wt % to about 10 wt %, or from about 0.5 wt % toabout 10 wt %, or from about 1 wt % to about 10 wt % of the totalcomposition.

Other non-ionic hydrophilic material such as copolymers of PEG and PPG(polypropylene glycol), e.g., poloxamers, can also be used. In oneexample, Poloxamer-188 (which has an average molecular weight of about8400 Daltons) can be used.

In some embodiments, the composition further comprises pharmaceuticalcarriers or excipients compounds or materials which enable thecompositions to be presented in topically administrable semi-solidaqueous gel forms. For example, carboxymethylcellulose can be used as agel-forming agent. However, other cellulose derivatives such asmicrocrystalline cellulose as well as polysaccharides such as alginateand agarose, tragacanth, guar gum, xanthum gum, are also suitable asgel-forming agents. The gel may, if required, be made thicker and/orstiffer by addition of a relatively resilient gel-forming material suchas a cross-linked fibrous protein, e.g. gelatin or collagen cross-linkedwith formaldehyde.

In some embodiments, the composition can be in a form of a cream, whichcan include those excipients suitable for a cream formulation, such asparaffin oil, vaseline, wax, organic esters such as cetyl palmitate,etc.

In some embodiments, the composition of the invention further comprisesa thickener for desired viscosity of the composition for skin delivery.For example, the thickener can include hydroxypropyl cellulose (HPC).HPC can make the composition into a smooth film for easy application. Italso reduces evaporation and allows the wound to stay moist longer, afactor that has been shown to improve healing and result in decreasedscarring. There are different grades of HPC available according tomolecular weights or viscosity of certain concentrations of HPC watersolution.

In some embodiments of the composition, XAV939 can constitute from about0.001 wt % to about 5 wt % of the total composition (including water).In some embodiments, XAV939 can constitute from about 0.01 wt % to about2 wt %, from about 0.02 wt % to about 1 wt %, or from about 0.05 wt % toabout 0.5 wt % of the total composition. In some embodiments, GO-HAconstitutes from about 0.001 wt % to about 5 wt % of the totalcomposition. In some embodiments, GO-HA can constitute from about 0.01wt % to about 2 wt %, from about 0.02 wt % to about 1 wt %, or fromabout 0.05 wt % to about 0.5 wt % of the total composition.

In the compositions as described herein, other pharmaceutical ortherapeutic compounds may be included in addition, or as an alternative,to XAV939. In other words, the compositions with XAV939 present or withXAV939 removed can also serve as a base dispersion medium in which otherpharmaceutical or therapeutic agents, especially those which arehydrophobic, may be dispersed, e.g., for topical administration to treatwound. These agents may include antifibrotic compounds such aspirfenidone, halofuginone, nintedanib, tocilizumab, rilonacept, etc.,anti-cancer agents, anti-inflammatory agents, analgesics, antibiotics,etc.

In another aspect of the invention, a medical device is provided, whichincludes the composition as described herein, and a substrate upon whichthe composition is applied. The medical device can be in a form thatfacilitates topical administration of the composition, where thesubstrate can be constructed with suitable strength and flexibility forcovering, securing and/or protecting the wound. For example, thesubstrate can be a patch, a pad, a tape, a bandage, a gauze, a suture,etc.

In yet another aspect of the invention, a method of treating a cutaneousor dermal wound in a subject (e.g., a human or a non-human animal) isprovided. The method comprises contacting the wound with an effectiveamount of the composition as described herein. The wound can be a typewhere its normal healing is accompanied by scar formation. The wound canbe a surgical wound that is caused by a physical impact that disruptsthe structure and function of the skin (such as a laceration, abrasion,cut, scratch or puncture by a knife, scalpel, bullet, or other sharp orblunt objects). The wound can also be caused by excessive (low or high)temperature, such as a burn. The wound can also be a chronic wound thatdoes not heal in expected time due to the lack of one or more of themain requirements of healing, including a good supply of blood, oxygenand nutrients, and a clean and infection-free environment. Examples ofchronic wounds include ischemic wounds where the wound area is notgetting sufficient blood supply. Diabetic ulcers are a common type ofischemic wounds.

The composition(s) of the present invention described herein can beadministered by applying the composition(s) topically on the wound site.If the composition is included in a medical device described hereinwhich includes a substrate such as a patch or a pad, the medical devicecan be secured to the wound site such that the composition contacts thewound.

In some embodiments, the method of treatment can include delivering asecond wound medication or therapeutic agent to the subject, comprisingone or more of: corticosteroid, a cytotoxic drug, an antibiotic, anantiseptic, nicotine, an anti-platelet drug, an NSAID, colchicine, ananti-coagulant, a vasoconstricting drug or an immunosuppressive, agrowth factor, an antibody, a protease, a protease inhibitor, anantibacterial peptide, an adhesive peptide, a hemostatic agent, livingcells, honey, or nitric oxide. These therapeutic agents can be deliveredas separate dosage forms from the compositions described herein, or maybe included as additional components of the compositions describedherein, hence delivered together with XAV939.

In a further aspect, the present invention provides methods forpreparing the compositions described herein and the intermediatecompounds. In one embodiments, a method of preparing a GO-HA conjugateis provided, which includes: (a) modifying GO by converting at leastsome of the benzoxylic acid groups of the graphene oxide to terminalaliphatic carboxylic acid groups; (b) derivatizing HA by reacting HAwith a reagent having dual functional groups reactive to the terminalaliphatic carboxylic acid groups, the dual functional groups beingintervened by a spacer group; and (c) reacting the modified GO obtainedin (a) and derivatized HA obtained in (b), thereby forming a GO-HAconjugate.

In the above preparation method, the spacer group can be anunsubstituted or substituted, saturated or unsaturated linear alkylenegroup having 2-20 backbone carbons. For illustration and not limitation,the reagent for derivatizing HA can be selected from the following:

where R¹ and R² can be independently —CONHNH₂, —SH, —NH₂, —OH, or othernucleophiles, and n is an integer and can be for example, 1-20, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, etc. In some embodiments, the reagent forderivatizing HA can be a dihydrazide, such as adipic acid dihydrazide.

In some embodiments, a method for preparing a composition of the presentinvention includes: obtaining GO-HA (e.g., by the methods above),dissolving the GO-HA conjugate in water to obtain a GO-HA watersolution, and adding XAV939 to the GO-HA water solution to form amixture. In some examples, this is accomplished by dissolving XAV939first in a non-ionic hydrophilic polymer, e.g., PEG-400 (or PEG 400,having an average molar mass of about 400), and then the XAV939 solutionis added into the GO-HA conjugate water solution.

The present invention provides a wound treatment strategy thatsimultaneously addresses several facets of wound healing bysynergistically combining a multi-functional scaffold (GO) conjugated toa highly hygroscopic material (HA) traditionally beneficial in woundhealing, and aqueous solubilization of a potent Wnt pathway inhibitor(XAV939) for improved healing of cutaneous wounds with reduced scarring.Without wishing to be bound by any particular theory, it is believed thecompositions and methods of the present invention can prevent, reduce,or inhibit dermal fibrosis and scarring in wound healing by inhibitingTNK1 and 2 via XAV939 targeting the Wnt/β-catenin in the skin, therebydirecting wound healing toward a regenerative process rather than afibrotic process. It is believed that XAV939 is coated onto the GO as ananocarrier due to the ability of GO to complex (via π-π interactions)hydrophobic compounds, and with the hydrophilic HA linked to GO,rendering the hydrophobic compounds “water soluble”. Also, GO may serveas a scaffold for cell growth and communication due to its goodbiocompatibility, and properties that influence cell-cell-communication,cell division and cell fate, as well as possible suppression ofmicrobes.

Additional benefits of the topical composition and topicaladministration of embodiments of the present invention is low toxicityand high bioavailability of the beneficial components to the site ofinjury/wound.

The following examples are provided for purpose of illustration ofcertain aspects of the description herein and should not be deemed tolimit the invention in any way.

The instrumentation used in the Examples: FT-IR: Thermo Nicolet 380FT-IR with a SmartOrbi Diamond ATR accessory; 1H NMR: 500 MHz BrukerDRX500 or AV-500 NMR spectrometer; UV-Vis: Shimadazu Pharma Spec UV1700.Solvents: 99% pure, supplied by Sigma-Aldrich; Sonication was conductedat 42 kHz in a bath sonicator.

Example 1: Modification of GO

Although GO includes many carboxylic acid groups on its edges, thereactivity of these aromatic carboxylic acid groups is not high. Toimprove its linking efficiency with HA, some of these aromaticcarboxylic acid groups on GO are converted to aliphatic aromaticcarboxylic groups. See Scheme 1. In this step, some hydroxyl groups ofGO are also converted to carboxylic groups (as illustrated above).

Graphene oxide (1.25 g, 250 mL of 5 mg/mL GO dispersion in de-ionizedwater; supplier: Goographene Inc.) was added to 250 mL of ultra-purede-ionized water and stirred for 5 minutes. Sodium hydroxide pellets (3g (0.075 moles; supplier: Sigma-Aldrich) were added in small solidportions to the mixture over 30 minutes. Once addition was complete, itwas stirred for 1 hour at room temperature. Next the solution wasultrasonicated for 30 minutes, and then chloroacetic acid (3.54 g(0.0375 moles); supplier: Alfa Aesar) was added in small, solid portionsover 20 minutes. The reaction mixture was then stirred for 18 hours atroom temperature. The reaction mixture was acidified with hydrochloricacid (7 mL, 12N). The solution was then transferred to centrifuge tubesand centrifuged for 15 minutes at 5,000 rpm. The water layer was thendecanted and more ultra-pure deionized water (˜30 mL) added to the tubesbefore re-centrifugation. This process was repeated 3 times. Methanol(˜30 mL) was added to the precipitated, modified graphene oxideremaining in the centrifuged tubes and centrifuged at 5,000 rpm for 15minutes. This process was repeated 3 times. Once the methanol wasdecanted, the tubes were put under vacuum for 48 hours at roomtemperature for drying. A total of 0.926 g of modified graphene oxidewas obtained. ¹H NMR (500 MHz, D₂O) δ: 4.173 (—CH₂CO₂H) ppm (diagnosticpeak). FT-IR: 1593 cm⁻¹, (C═C), UV-Vis λ: 268 nm.

Example 2: Derivatization of HA

The general procedure of derivatizing HA is shown in Scheme 2.Hyaluronic acid (100 mg, MW=10,000 (n=13.5 in the scheme), 0.00001moles; supplier: Creativepegworks) in ultra-pure deionized water (20 mL)was stirred for 5 minutes at room temperature.1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) (52mg, 0.00027 moles, supplier: Alfa Aesar) was added to the mixture andstirred for 3 hours at room temperature. During this time, the pH of thesolution was maintained at approximately 5 to 6 by small additions of0.1N hydrochloric acid. This mixture was added dropwise to a separatemixture containing adipic dihydrazide (ADH) (17 mg, 0.00027 moles;supplier: Alfa Aesar) in 5 mL of ultra-pure deionized water at roomtemperature. Once the addition was complete, it was stirred at roomtemperature for 18 hours. The solution was then subjected to dialysis(MWC=3500) for 24 hours. The mixture was then lyophilized to obtain awhite powder (100 mg). ¹H NMR (500 MHz, D₂O) δ: 2.4 (2H), 2.26 (2H),1.66 (4H) ppm (diagnostic peaks). The substitution/loading degree wasdetermined by the ratio of methylene hydrogens of adipic hydrazide toacetyl methyl protons of the Hyaluronic acid moiety. Integrationindicated 30% coupling, resulting in approximately 8 substitutions.Coupling ranges were commonly 6-30%. A 6% coupling resulted in ˜1.6carboxyl units substituted.

Example 3: Reaction Between Modified GO and Derivatized HA to PrepareGO-HA Conjugate

The general procedure of preparing GO-HA is outlined in Scheme 3 below.The modified GO obtained in Example 1 (0.90 g) was added to ultra-puredeionized water (100 mL) and stirred for 5 minutes, followed byultra-sonication for 15 minutes.2-Succinimido-1,1,1,3-tetramethyluronium tetrafluoroborate (TSTU) (0.32g, 0.001 moles; supplier: Alfa Aesar),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate,Hexafluorophosphate Benzotriazole Tetramethyl Uronium (HBTU) (0.41 g,0.001 moles; supplier: Aldrich) and diisopropylethyl amine (DIEA) (0.15mL, 0.001 moles; supplier: Sigma-Aldrich) was added to the mixture atroom temperature and stirred for 5 minutes. Then a solution ofhyaluronic acid-adipic hydrazide (obtained from Example 2) (0.90 g in150 mL of ultra-pure deionized water) was added dropwise to theactivated graphene oxide solution at room temperature. The reactionmixture was allowed to stir for 18 hours and then subjected to dialysis(MWC=20,000) for 2 days. The solution was then lyophilized to yield ablack powder (1.13 g). ¹H NMR (500 MHz, D₂O) δ: 1.94 (2H), 1.90 (2H),1.21 (4H) ppm (diagnostic peaks).

Example 4: Preparation of Wound-Treating Composition

The procedure of preparing the composition is schematically shown inScheme 4. 11 mg of the GO-HA obtained in Example 3 was dissolved in 11mL of ultra-pure water, to create an effective concentration of 1 mg/mL.The solution was ultrasonicated for 10 minutes. XAV939 (11 mg; supplier:APEBIO) was added to PEG-400 (0.5 mL) and subjected to ultrasonicationfor 30 minutes. The XAV939 PEG-400 solution was added dropwise to theGO-HA and vigorously stirred for 5 minutes. The combined solution wasthen subjected to ultrasonication for 1 hour and then stirred at roomtemperature for 18 hours. Hydroxypropyl cellulose (0.2 g; supplier:Sigma-Aldrich) was added in small portions at room temperature withvigorous stirring. Once addition was complete, the solution was stirredat room temperature for 24 hours to form a viscous solution of theGO-HA/XAV939 complex. The complex was to be used as is.

Example 5. Animal Study 1

The objective of this study was to observe the wound healing effect of acomposition of the present invention (STM42) in a dermal full thicknessinjury rat model following topical administration.

5-1: Test Article

STM42: the composition made according to the procedure described inExample 4.

Vehicle: 96 vol % water and 4 vol % PEG-400.

5-2 Animal Husbandry

16 Sprague-Dawley rats at age of about 6-8 weeks were used. Animal roomwas set to maintain a temperature of 23±2° C., humidity of 40-70%, and a12-hour light/12-hour dark cycle. SPF Rat Growth Breeding Feed wasprovided ad libitum throughout the in-life portion of the study. ReverseOsmosis water was available to the animals ad libitum. Animals were freeto access both food and water during the whole course of study.

5-3 Animal Grouping and Test Procedure

Animals were housed 5 rats per cage and acclimated for 5-7 days. Theywere then each anesthetized with pentobarbital sodium (45 mg/kg, ip., 2%in saline) and shaved on both sides of the back with an electricclipper, disinfected by applying betadine followed by 70% ethanolwiping. A (3 to 4 cm²) full thickness of elliptical excision wound wascreated using toothed forceps, Acupunch® (12 mm, Acuderm Inc, USA) andscissors, skin was removed from the underlying muscle.

The animals were allocated to two groups according to the wound areasand body weight. Each group consists of 8 animals. Vehicle group: G1(G1-1, G1-2, G1-3, G1-4, G1-5, G1-6, G1-7, G1-8); STM42 group: G2 (G2-1,G2-2, G2-3, G2-4, G2-5, G2-6, G2-7, G2-8). The grouping is summarizedbelow.

Number of Dosage Route of Dosing Group animals Treatment (μL/rat) Adm.Schedule 1 8 Vehicle 100 Topical QD × 14 d 2 8 STM42 100 Topical QD × 14d

STM42 and vehicle were topically administered to the animalsrespectively via a syringe to drip the formulation directly into thewound bed to create an even and consistent layer over the wound to coverall of the wound area. The treatment was continued daily for 2 weeks (14days) following injury.

The animal body weight was measured twice weekly. Wound area boundarywas drawn on a transparency paper with a permanent marker on Day 1, Day5, Day 10 and Day 15 and the area will be measured with ImageProPremier®. Photos of the wound of each rat were taken on Day 1, Day 5,Day 10 and Day 15.

For HE staining: Two 5 um slides per wound was stained with H&E andMasson's Trichrome and digitally scanned for pathology analysis. Thepathology analysis included: (1) Evaluation of Scar resolution and reteridges formation (dermal-epidermal junctions) in healing skin: CreateMassons trichrome stained sections of skin from the full-thicknessexcisional wounds and evaluate “linear” extracellular matrix (scar-like)for all articles. (2) Evaluation of fiber thickness of healing skin byMassons trichrome stained sections imaged with circular polarized lightfor each article. (3) Evaluate rete-ridge for each article by H&Estaining for each article.

5-4 Pathology Testing:

Materials: Skin samples, 10% formalin, hematoxylin, Eosin stainingsolution, acid fuchsin, Aniline Blue, dimethylbenzene, 70% alcohol, 95%alcohol, 100% alcohol, etc.

Instruments: Tissue hydroextractor (Shandon Excelsior ES™ TissueProcessor Fisher/Thermo A78400006); Tissue Embedder (ShandonHistocentre™ 3 Tissue Embedding Center Fisher/Thermo B64100010); Leicaautomatic slicing machine (Leica RM2255); Automatic staining machine(Automatic Slide Stainer Fisher/Thermo A74200010, Shandon Varistain®24-4)

Tissue processing & slicing: (1) Tissue processing by tissuehydroextractor: gradient alcohol dehydration, transparentizing bydimethylbenzene, paraffin infiltration, embedding. (2) The samples arecut into 5 μm thick slides by Leica automatic slicing machine (3) H&Estaining, Masson staining.

Evaluation parameters: re-epithelialization, neovascularization, ReteRidge, scabbing, granulation. Scoring criteria are summarized in thebelow table:

1. Re-epithelialization New epithelium 0 No ingrowth of epithelium intothe wound gap attempting to bridge 1 Small stumps of growth from theedge over the wound gap 2 Large gap between the bridging epithelium 3Small gap between the bridging epithelium 4 Complete bridging over thewound 2. Neovascularization Newly formed 0 Relatively normal vascularnetwork in 1 Mildly increased in number in the wound bed the wound bed 2Moderately increased in number in wound bed 3 Moderately to markedlyincreased in number in wound bed 4 Markedly increased in number in woundbed 3. Rete-ridge Newly formed 0 No formation of rete ridge columns of 1Few columns formed epithelium 2 Mildly increased in number extending 3Moderately increased in number downward to dermis 4 Markedly increasedin number of the wound bed 4. Scabbing Formation of scab 0 Relativelynormal on the cornified 1 Small amount of scabbing layer or on the 2Moderate amount of scabbing wound bed 3 Moderate to marked amount ofscabbing 4 Marked amount of scabbing 5. Granulation Accumulation of 0Relatively normal inflammatory cells 1 Small amount scattered in thewound bed in the matrix of 2 Moderate amount across the wound bedfibrotic/scar tissues 3 Dense fibrotic tissues with heavy infiltrationof in the wound bed of inflammation dermis 4 Fibrinoid formation in thedermis

5-5 Observation & Analysis

Clinical signs: all clinical signs were recorded for individual animals,once before commencement of treatment and once daily during the study;observations were performed at the same time interval each day.

Terminal Studies: Animals in extremis or disposed for humane reasons andthose that have completed the scheduled test period would be euthanizedby carbon dioxide.

Statistical Analysis: The results (individual and group) were analyzedusing Student's unpaired t-test. Data were given as Mean±SD or Mean±SEM.P<0.05 was considered significant.

5-6 Results

Clinical signs: Mortality, morbidity and the abnormal behavior were notfound during the treatment period of the experiment. During the courseof the experiment, the following signs did not occur: suffering(cachexia, weakening, difficulty to move or to eat, pain, cryings);toxicity (hunching, convulsions); 25% body weight loss for threeconsecutive days or 20% body weight loss on any day. There was nosignificant difference between groups in wound areas during theexperiment (p>0.05).

On Day 15, 3 rats in each group (vehicle group: G1-4, G1-5, G1-8; STM42group: G2-3, G2-4, G2-6) with an average wound healing rate wereeuthanized with carbon oxide, and wound tissue were excised and fixedneutral formalin buffer then embedded in paraffin for histology.

The scores of the STM42 group and the vehicle group are represented inFIG. 1. From FIG. 1 it can be seen that STM42 had a significant effecton re-epithelization (essential component of wound healing used as adefining parameter of a successful wound closure), improvedneovascularization (the natural formation of new blood vessels, usuallyin the form of functional microvascular networks, capable of perfusionby red blood cells, that form to serve as collateral circulation inresponse to local poor perfusion or ischemia), improved rete ridgeformation (epithelial extensions that project into the underlyingconnective tissue in both skin and mucous membranes-improved rete ridgeformation is a sign of tissue regeneration rather than fibrosis/scarring(reduced rete ridge formation)), decreased scabbing (wounds with scabstake longer to heal and a sign of fibrosis/scarring) and decreasedgranulation (granulation tissue is composed of extracellular matrixproteins such as fibrin and type III collagen that can be quickly laiddown by the initial surge of fibroblasts that is recruited to the woundsite-causing a scar).

The total effect of STM42 toward wound healing with scar reduction canbe evaluated using the following formula:Total=(Re-epithelization+Neovascularization+Rete ridgeformation)−(Scabbing+Granulation)). Using mean measured values for eachcategory: STM42=4.66. Vehicle=−1.33. Under this metric, there is a5.99-fold increase in scar reduction using STM42 in this study ascompared to the control group.

Example 6. Animal Study 2

The objective of this study was to observe the wound healing effect ofSTM-52 in a dermal full thickness injury STZ induced diabetic rat modelwith topical administration.

6-1 Compound and Preparation

STM-52 (or STM52): same as STM42, the composition made according to theprocedure described in Example 4.

Vehicle: 96 vol % water and 4 vol % PEG-400.

6-2 Test Article Preparation Procedure

Admixing the STM-52 well by sonicating for 10 minutes before injectingvia syringe 100 uL of sample onto the wound.

6-3 Animal Husbandry

14 male Spargue-Dawley rats, grade SPF of an age of about 6-8 weeks wereused. The rats had a blood glucose of >16.7 mM.

Weight at initiation of treatment: Within the range of 200-300 g.

Acclimation period: 7 days.

Environmental controls for the animal room were set to maintain atemperature of 23±2° C., humidity of 40˜70%, and a 12-hour light/12-hourdark cycle. The 12-hour dark cycle may be temporarily interrupted toaccommodate study procedures.

Food and water: SPF Rat Growth Breeding Feed (BEIJING KEAO XIELI FEEDCO. LTD.) was provided ad libitum throughout the in-life portion of thestudy. Reverse Osmosis water was available ad libitum.

Animal selection and fasting: Animals to be used in this study wereselected based on overall health and acclimation to caging. Animals werefree to access both food and water during the whole course of study.

6-4 Experimental Design

Upon arrival at the animal facility, the animals were housed 5 rats percage and acclimated for seven days. Prior to the experiments, food waswithdrawn for about 16 hours. The rats were rendered diabetic by an i.p.injection of STZ (65 mg/kg) dissolved in sodium citrate buffer, pH 4.5.The rats with a final blood glucose level>16.7 mM (300 mg/dL) wereincluded in the study. All the selected rats were anesthetized withpentobarbital sodium (45 mg/kg, i.p. 2% in saline) and shaved on bothsides of the back with an electric clipper, disinfected by iodophorfollowed by 75% ethanol wiping. A (3 to 4 cm²) full thickness ofelliptical excision wound was created using Acupunch® (12 mm, AcudermInc, USA). The skin was removed from the underlying muscle usingscissors and toothed forceps.

The animals were divided into two groups according to the wound area andthe body weight.

Number of Dosage Route of Group Animals Treatment (μL/rat) Adm. DosingSchedule 1 7 Vehicle 100 Topical QD × 21d 2 7 STM-52 100 Topical QD ×21d

The wound was topically administrated (via a syringe to drip theformulation directly into the wound bed to create an even and consistentlayer over the wound making sure to cover all of the wound area) withSTM-52 or vehicle (100 μL/rat). The treatment was continued daily for 21days following injury.

FIG. 2 is a plot showing the average area size of the wounds of theVehicle group and the STM-52 group during the course of the 21-daytreatment. The results indicate that topical administration of STM-52increased the rate of healing of the diabetic rat impaired healinganimal model as compared to vehicle. Primarily, the rate increase wasobserved on days 10-18 of the 21 day study. Also during this time, therewere no signs of overt toxicity as indicated by body weightmeasurements.

The word “about” as used herein in association with a numeric value or anumeric range mean “approximately” and refers to a result that can beobtained within a tolerance and the skilled person knows how to obtainthe tolerance, for example, ±10% of the given value or range.

The term “effective amount” as used herein means the amount of acomposition that, when administered to a subject for treating anundesirable or diseased condition (e.g., treating a wound) of thesubject, is sufficient to ameliorate or improve such condition. The“effective amount” may vary depending on the composition, the conditionand its severity, and the age, physical condition, and responsiveness ofthe subject to be treated.

While various inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, composition, device, system, article, material, kit, and/ormethod described herein. In addition, any combination of two or moresuch features, compositions, devices, systems, articles, materials,kits, and/or methods, if such features, compositions, devices, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

Also, various inventive concepts may be embodied as one or more methods,of which an example has been provided. The acts performed as part of themethod may be ordered in any suitable way. Accordingly, embodiments maybe constructed in which acts are performed in an order different thanillustrated, which may include performing some acts simultaneously, eventhough shown as sequential acts in illustrative embodiments.

1. A composition for treating a wound, comprising: a matrix componentcomprising a graphene oxide (GO) and hyaluronic acid (HA) conjugate(GO-HA), wherein the GO and HA are covalently linked via a linker;XAV939; and water.
 2. The composition of claim 1, further comprisingpolyethylene glycol (PEG).
 3. The composition of claim 2, wherein thePEG has a molecular weight of from about 200 to about 400 Daltons. 4.The composition of claim 2, wherein the PEG is in an amount of fromabout 0.1 wt % to about 20 wt % of the total composition.
 5. Thecomposition of claim 1, further comprising a thickener.
 6. (canceled) 7.The composition of claim 1, wherein the linker comprises 2-25 carbons.8. (canceled)
 9. The composition of claim 1, wherein the linkercomprises one or more —CH₂CH₂O-units.
 10. The composition of claim 1,wherein the linker comprises —R^(x)—R^(S)—R^(y)—, wherein R^(x) andR^(y) are each independently selected from the group consisting of —CO—,—COO—, —NH—, —NH—NH—, —NH—NH—CO—, —CS—, —S—, and —O—, and wherein R^(S)is an unsubstituted or substituted linear alkylene group having 2-20backbone carbons.
 11. The composition of claim 10, wherein R^(x) andR^(y) are each —NH—NH—CO—.
 12. The composition of claim 1, wherein theweight ratio of XAV939 to GO-HA is from about 1:2 to about 2:1.
 13. Thecomposition of claim 1, wherein XAV939 constitutes from about 0.001 wt %to about 5 wt % of the total composition.
 14. The composition of claim1, wherein the GO-HA constitutes from about 0.001 wt % to about 5 wt %of the total composition.
 15. A medical device comprising: a substrate;and the composition of claim 1 applied on the substrate.
 16. The medicaldevice of claim 15, wherein the substrate is a patch, a pad, a suture, agauze, a tape, or a bandage.
 17. A method of treating a cutaneous woundin a subject, comprising contacting the wound with an effective amountof the composition of claim
 1. 18. The method of claim 17, wherein thewound is a surgical wound.
 19. The method of claim 17, wherein the woundis a burn.
 20. The method of claim 17, wherein the wound is a chronicwound.
 21. (canceled)
 22. The method of claim 17, wherein the subject isa human. 23.-27. (canceled)